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Characterization of the β-glucuronidase Pn3Pase as the founding member of glycoside hydrolase family GH169
Glycobiology ( IF 4.3 ) Pub Date : 2020-07-28 , DOI: 10.1093/glycob/cwaa070
Paeton L Wantuch 1, 2 , Satya Jella 2 , Jeremy A Duke 1, 2 , Jarrod J Mousa 3, 4 , Bernard Henrissat 5, 6, 7 , John Glushka 8 , Fikri Y Avci 1, 2
Affiliation  

Abstract
Paenibacillus sp. 32352 is a soil-dwelling bacterium capable of producing an enzyme, Pn3Pase that degrades the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (Pn3P). Recent reports on Pn3Pase have demonstrated its initial characterization and potential for protection against highly virulent S. pneumoniae serotype 3 infections. Initial experiments revealed this enzyme functions as an exo-β1,4-glucuronidase cleaving the β(1,4) linkage between glucuronic acid and glucose. However, the catalytic mechanism of this enzyme is still unknown. Here, we report the detailed biochemical analysis of Pn3Pase. Pn3Pase shows no significant sequence similarity to known glycoside hydrolase (GH) families, thus this novel enzyme establishes a new carbohydrate-active enzyme (CAZy) GH family. Site-directed mutagenesis studies revealed two catalytic residues along with truncation mutants defining essential domains for function. Pn3Pase and its mutants were screened for activity, substrate binding and kinetics. Additionally, nuclear magnetic resonance spectroscopy analysis revealed that Pn3Pase acts through a retaining mechanism. This study exhibits Pn3Pase activity at the structural and mechanistic level to establish the new CAZy GH family GH169 belonging to the large GH-A clan. This study will also serve toward generating Pn3Pase derivatives with optimal activity and pharmacokinetics aiding in the use of Pn3Pase as a novel therapeutic approach against type 3 S. pneumoniae infections.


中文翻译:

β-葡萄糖醛酸酶 Pn3Pase 作为糖苷水解酶家族 GH169 创始成员的表征

摘要
类芽孢杆菌 32352是一种土壤细菌,能够产生一种酶 Pn3Pase,该酶可降解肺炎链球菌血清型 3 (Pn3P) 的荚膜多糖。最近关于 Pn3Pase 的报告已经证明了它的初步特征和保护对抗高毒性肺炎链球菌的潜力血清型 3 感染。最初的实验表明这种酶作为外切-β1,4-葡糖醛酸酶来切割葡糖醛酸和葡萄糖之间的 β(1,4) 键。然而,这种酶的催化机制仍然未知。在这里,我们报告了 Pn3Pase 的详细生化分析。Pn3Pase 与已知的糖苷水解酶 (GH) 家族没有显着的序列相似性,因此这种新型酶建立了一个新的碳水化合物活性酶 (CAZy) GH 家族。定点诱变研究揭示了两个催化残基以及截断突变体,这些突变体定义了功能的基本结构域。筛选 Pn3Pase 及其突变体的活性、底物结合和动力学。此外,核磁共振波谱分析显示 Pn3Pase 通过保留机制起作用。本研究在结构和机制水平上展示了 Pn3Pase 活性,以建立属于大型 GH-A 家族的新 CAZy GH 家族 GH169。该研究还将有助于生成具有最佳活性和药代动力学的 Pn3Pase 衍生物,以帮助将 Pn3Pase 用作针对 3 型的新型治疗方法肺炎链球菌感染。
更新日期:2020-07-28
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